Herpes Simplex Virus 1 pUL34 Plays a Critical Role in Cell-to-Cell Spread of Virus in Addition to Its Role in Virus Replication

Department of Microbiology, The University of Iowa, 3-432 Bowen Science Building, Iowa City, IA 52242, USA.
Journal of Virology (Impact Factor: 4.44). 07/2011; 85(14):7203-15. DOI: 10.1128/JVI.00262-11
Source: PubMed


Herpes simplex virus (HSV) pUL34 plays a critical role in virus replication by mediating egress of nucleocapsids from the
infected cell nucleus. We have identified a mutation in pUL34 (Y68A) that produces a major defect in virus replication and
impaired nuclear egress but also profoundly inhibits cell-to-cell spread and trafficking of gE. Virion release to the extracellular
medium is not affected by the Y68A mutation, indicating that the mutation specifically inhibits cell-to-cell spread. We isolated
extragenic suppressors of the Y68A plaque formation defect and mapped them by a combination of high-throughput Illumina sequencing
and PCR-based screening. We found that suppression is highly correlated with a nonsense mutation in the US9 gene, which plays
a critical role in cell-to-cell spread of HSV-1 in neurons. The US9 mutation alone is not sufficient to suppress the Y68A
spread phenotype, indicating a likely role for multiple viral factors.

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    • "However, either the NEC or its components may play roles in herpesvirus replication beyond nuclear egress. Thus, mutations have been identified in pUL34 which affect viral cell-to-cell spread and trafficking of glycoprotein E (Haugo et al., 2011). Conversely, mutations have been described which do not support efficient nuclear egress but partially restore plaque formation (Schuster et al., 2012). "
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    ABSTRACT: Herpesvirus capsids are assembled in the nucleus of infected cells whereas final maturation occurs in the cytosol. To access the final maturation compartment, intranuclear capsids have to cross the nuclear envelope which represents a formidable barrier. They do so by budding at the inner nuclear membrane, thereby forming a primary enveloped particle residing in the perinuclear cleft. Formation of primary envelopes is driven by a heterodimeric complex of two conserved herpesviral proteins, designated in the herpes simplex virus nomenclature as pUL34, a tail-anchored transmembrane protein located in the nuclear envelope, and pUL31. This nuclear egress complex (NEC) recruits viral and cellular kinases to soften the nuclear lamina and allowing access of capsids to the NEC. How capsids are recruited to the budding site and into the primary virus particle is still not completely understood, nor is the composition of the primary enveloped virion in the perinuclear cleft. Fusion of the primary envelope with the outer nuclear membrane then results in translocation of the capsid to the cytosol. This fusion event is clearly different from fusion during infectious entry of free virions into target cells in that it does not require the conserved essential core herpesvirus fusion machinery. Nuclear egress can thus be viewed as a vesicle (primary envelope)-mediated transport of cargo (capsids) through the nuclear envelope, a process which had been unique in cell biology. Only recently has a similar process been identified in Drosophila for nuclear egress of large ribonucleoprotein complexes. Thus, herpesviruses appear to subvert a hitherto cryptic cellular pathway for translocation of capsids from the nucleus to the cytosol.
    Preview · Article · Oct 2012 · Cellular Microbiology
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    ABSTRACT: Late in infection herpesviruses move DNA-filled capsids from the nucleus to the cytoplasm by enveloping DNA-containing capsids at the inner nuclear membrane (INM) and deenveloping them at the outer nuclear membrane. This process requires two conserved herpesvirus proteins, pUL31 and pUL34. Interaction between pUL34 and pUL31 is essential for targeting both proteins to the nuclear envelope (NE), and sequences that mediate the targeting interaction have been mapped in both proteins. Here, we show that a mutation in the INM-targeting domain of pUL34 fails to support production of infectious virus or plaque formation. The mutation results in multiple defects, including impaired interaction between pUL34 and pUL31, poor NE targeting of pUL34, and misregulated, capsid-independent budding of the NE. The mutant defects in virus production, plaque formation, and pUL31 interaction can be suppressed by other mutations in the INM-targeting domain of pUL31 and by additional mutations in the pUL34 coding sequence.
    Preview · Article · Sep 2011 · Journal of Virology
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    ABSTRACT: Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.
    Preview · Article · Dec 2011 · Journal of Virology
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